Ribosome inactivating proteins (RIPs) have been used as instruments of biological warfare and terrorism. Ricin is a heterodimeric plant toxin that consists of A and B-chains and the prototype of type II RIPs. Its B- chain is a lectin that enables cell binding. After endocytosis, the A-chain translocates through the endoplasmic reticulum membrane to reach the cytosol where its N-glycosidase activity removes a specific adenine from the highly conserved, sarcin/ricin loop (SRL) in the large rRNA. Ricin has been classified as Category B priority for biodefense. Very little is known about the membrane translocation and ribosome interactions of ricin and the molecular mechanism by which it causes apoptosis in mammalian cells. We have established yeast as a biologically relevant model system to study the activity of RTA, and isolated mutant forms of RTA, which do not kill cells. Translation inhibition by ribosome depurination has been hypothesized to be responsible for the cytotoxicity of ricin. However, our preliminary analysis of the nontoxic RTA mutants indicates that ribosome depurination is necessary, but not sufficient for cytotoxicity. We further showed that wild type RTA inhibits induction of the unfolded protein response (UPR) in yeast and in mammalian cells and induces activation of JNK, p38 and ERK signaling pathways in mammalian cells. This project aims to use yeast and mammalian cells as complementary systems to understand the molecular basis for ricin intoxication. Specific Aims: 1. Using the nontoxic RTA mutants, determine if ribosome binding and depurination are required for cytotoxicity in yeast and induction of apoptosis in mammalian cells. 2. Characterize the interaction between RTA and ribosomal protein POand determine if binding to POis essential for ribosome depurination by RTA. 3. Determine if RTA causes cell death by inhibiting the development of UPR in yeast and in mammalian cells. 4. Identify the cellular genes necessary for cytotoxicity of RTA in yeast. Ricin is not only a bioterrorism threat, but inhibits translation by a similar mechanism as the bacterial enterotoxins. Therefore, the studies outlined in this application will have important implications for the design of protection strategies against AB-toxins that are classified as high-risk candidates for bioterrorism.